Since the majority of gluconeogenic carbons are derived from phosphoenolpyruvate (PEP) via the Krebs TCA cycle, hepatic glucose production is also intimately linked to acetyl-CoA oxidation and energy production. Together, these biochemical pathways form a metabolic network, as depicted in FIG. 1, that is highly responsive to matching the external demand for glucose with the availability of glycogen, gluconeogenic precursors and energy. Measurements of carbon flux through this network typically combine an isotope dilution measurement of endogenous glucose production (GP) with additional tracer measurements of the contributing pathways.
As one example, GP in fasting humans has been determined by analysis of the 2H-enrichment at glucose C6 using the hexamethylenetetramine (HMT) method and mass spectrometry after infusion of [6,6-2H2] glucose at a known rate. It is possible to also give oral 2H2O and measure the contribution of gluconeogenesis to glucose production from 2H-enrichments at glucose C5 versus C2 after parallel selective degradations of plasma glucose and analysis by mass spectroscopy. However, the contributions of glycerol versus TCA cycle intermediates to gluconeogenesis cannot be differentiated by this method nor can 13C tracers be combined with 2H tracers using the HMT method.
13C tracers have also been used to measure metabolic flux through the network. Endogenous glucose production has been monitored with [U—13C6] glucose by following disappearance of the parent m+6 isotopomer from plasma glucose. Additional metabolic information has been derived from partially-labeled glucose molecules generated by recycling of 13C-label with this method. However, the extent to which the recycled label can be reliably analyzed and related to rates of gluconeogenesis and TCA cycle flux is questionable.
Others have used tracers like [3-13C] lactate that enter at the level of the TCA cycle along with a separate tracer for measuring endogenous glucose production. Neither [U—13C6]glucose or [3-13C]lactate tracers, however, can differentiate glucose produced from glycogen, glycerol or the TCA cycle.